research article a computational approach for predicting...

Research ArticleA Computational Approach for Predicting Role ofHuman MicroRNAs in MERS-CoV Genome

Md Mahmudul Hasan1 Rozina Akter2 Md Shahin Ullah3 Md Jaynul Abedin4

G M Ahsan Ullah5 and Md Zakir Hossain2

1 Institute of Biomedical Studies Baylor University Waco TX 76706 USA2BioMedNanoTech Inc 500 South University Avenue Suite 319 Little Rock AR 72205 USA3Biotechnology and Genetic Engineering Discipline Khulna University Khulna 9208 Bangladesh4Civil Engineering Department Bangladesh University of Engineering and Technology Dhaka 1000 Bangladesh5Environment Science Discipline Khulna University Khulna 9208 Bangladesh

Correspondence should be addressed to Md Zakir Hossain mzhceobiomednanotechcom

Received 27 June 2014 Revised 24 September 2014 Accepted 27 September 2014 Published 23 December 2014

Academic Editor Huixiao Hong

Copyright copy 2014 Md Mahmudul Hasan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The new epidemic Middle East Respiratory Syndrome (MERS) is caused by a type of human coronavirus called MERS-CoVwhich has global fatality rate of about 30 We are investigating potential antiviral therapeutics against MERS-CoV by using hostmicroRNAs (miRNAs) which may downregulate viral gene expression to quell viral replication We computationally predictedpotential 13 cellular miRNAs from 11 potential hairpin sequences of MERS-CoV genome Our study provided an interestinghypothesis that those miRNAs that is hsa-miR-628-5p hsa-miR-6804-3p hsa-miR-4289 hsa-miR-208a-3p hsa-miR-510-3p hsa-miR-18a-3p hsa-miR-329-3p hsa-miR-548ax hsa-miR-3934-5p hsa-miR-4474-5p hsa-miR-7974 hsa-miR-6865-5p and hsa-miR-342-3p would be antiviral therapeutics against MERS-CoV infection

1 Introduction

The new epidemic Middle East Respiratory Syndrome(MERS) has emerged since recent years The first case ofMERS was reported at September 2012 in Saudi Arabia andseverity rate is increasing day by day [1] It is caused by atype of human coronavirus calledMERS-CoV a newmemberin the lineage C of 120573-coronavirus (120573-CoV) [2] So far (until15 July 2014) 688 patients have been infected by MERS-CoV globally where 30 people already died [1 3 4]Some recent studies [5 6] indicate that bats and dromedarycamels are potential host to transmit virus to human Theclinical symptoms of MERS-CoV are almost similar toSevere Acute Respiratory Syndrome Coronavirus (SARS-CoV) which emerged in 2003 [1] Although current severityrate of MERS-CoV is low this scenario could be changedrapidly globally We hope this study will play significantrole in order to develop a potential antiviral therapy againstMERS-CoV

Coronaviruses are positively scene enveloped singlestranded RNA viruses which encode 16 nonstructural pro-teins (nsps) including different essential and nonessentialproteins [7] However the replication mechanism of coron-avirus is still not fully clear but those proteins are thoughtto play vital role during viral life cycle as well as replicationSimilar to other RNAviruses coronavirus replicate in the hostcytoplasm The replication process is initiated by the viralparticle after binding with their specific cellular receptorsknown as S-protein mediated binding The positive strand ofRNA genome directly translated into replicase poly-proteinsand further cleaved by 16 nsp [7 8] So the study betweenhost and pathogen interaction always plays significant rolein order to develop potential antiviral therapeutics against allcoronavirus as well as MERS-CoV Although rational vaccinedesign is based on the neutralization activity of highly potentantibodies since discovery of miRNAs and RNAi many ofthe investigators reported miRNA mediated gene silencingactivity [9ndash11]

Hindawi Publishing CorporationAdvances in BioinformaticsVolume 2014 Article ID 967946 8 pageshttpdxdoiorg1011552014967946

2 Advances in Bioinformatics

MERS CoV (Strain Riad) complete genome

Hairpin like structure screen by VMir

74 miRNAs precursor candidate

Screen with all human miRNA by miRbase search

11 pre-miRNAs aligned with 13 human miRNAs

RNA hybrid web server to effective hybridization

RNAfold to predict secondary structure

Figure 1 Schematic representation of human miRNA prediction on MERS-CoV viral genome [27]

miRNAs are genomically encoded small noncoding RNAmolecule generally 19ndash26 base pairs in length which regulateposttranscriptional level genes expression [12ndash14] It is welldocumented that some plants animals and viruses encodethe miRNAs to regulate their diverse biological or physio-logical processes including development apoptosis tumor-ogenesis proliferation stress response and fat metabolism[15 16] Thus 30 424 mature microRNAs have been identifiedfrom 206 species where 2578miRNAs are encoded by humangenome [17] Virus encodedmiRNAs are unique because theyregulate not only their own gene expression but also their hostgene expression [18]

miRNA genes are transcribed by RNA polymerase IIand formed primary miRNA in nucleus Then primary miR-NAs cleaved into 60ndash90 base-pair-long hairpin intermediateknown as pre-miRNA by enzymatic activity of the RNaseIII ribonuclease Dicer [18ndash20] Pre-miRNAs are bound andexported from nucleus to cytoplasm by the action of enzymeexportin-5 and Ran (RAs-related Nuclear protein) [19] Inthe cytoplasm the pre-miRNAs are further cleaved by RNaseIII ribonuclease Dicer into a double stranded RNA knownas duplex mature RNA [19] Guided stand (active stand) ofduplex RNA is loaded to RNA-induced silencing complex(RISC) which targets messenger RNA to degrade or represstranslational activity [18] Perfect complementarity between31015840 untranslated region (UTR) of the mRNA and the seedregion of miRNA (2ndash7 bp) is sufficient result in cleavage butimperfect complementarity may block translation [18 21]Some recent study suggests that miRNA is being exploredas antiviral defense against several diseases including HIV-1 [22] HSV [23] Dengue [24] Influenza [21] and hepatitisC (HCV) [25] It has been reported that the use of miRNAsas an anti-HCV treatment demonstrated promising efficacy

and safety results in an early stage trial [26] In this study wecomputationally identified some potential targets of humanmicroRNA on Middle East Respiratory Syndrome Coron-avirus (MERS-CoV) genome Our study may help to betterunderstand host pathogen interaction as well as to developnew antiviral therapy against MERS-CoV

2 Materials and Methods

TheMERS-CoVmiRNA prediction was carried out using thecomplete genome sequence of MERS-CoV (GB KJ1569521)obtained from the National Center for Biotechnology Infor-mation (NCBI) Figure 1 shows a flowchart of the compu-tational prediction process [27] Briefly the viral genomewas scanned for hairpin-structured miRNA precursors usinga VMir Analyzer program [28 29] VMir is an ab initioprediction program which was designed specifically to iden-tify pre-miRNA in viral genome The scanned hairpins werevisualized in VMir viewer where 74 sequences with potentialhairpin like structures were extracted as candidate miRNAprecursor Each of the sequences of the candidate miRNAprecursors was searched for nucleotide similarity with allhuman microRNAs by using SEARCHmenu of the miRBasedatabase (httpwwwmirbaseorgsearchshtml) [17]

Then 11 sequences were identified as candidate miRNAprecursor based on significant sequence similarity withhuman miRNAs The hybridization between the viral pre-cursor miRNAs and complementary template of the poten-tial human miRNAs were analyzed by RNA hybrid webserver (httpbibiserv2cebitecuni-bielefelddernahybrid)[30] Finally the RNAfold web server (httprnatbiunivieacatcgi-binRNAfoldcgi) was used to predict the secondarystructure of pre-miRNA [31]

Advances in Bioinformatics 3

225

200

150

100

50

0

1 5000 10000 15000 20000 25000 30000

(a)

200

150

100

50

0

1 5000 10000 15000 20000 25000 30000

(b)

Figure 2 Graphical view of VMir analysis of the MERS-CoVgenome (a) All hairpins of pre-miRNAs were shown using defaultsettings The hairpin is plotted according to the positions of theviral genome (b) Customized view of predicted pre-miRNA afterfiltering (minimum hairpin size 60 nt maximum hairpin size120 nt minimum hairpin score 115 and minimum window count25)

3 Result

31 Prediction of Precursor miRNA (Pre-miRNA) Hairpinswith VMir MERS-CoV viral genome was screened withVMir Analyzer program and the result of VMir analyzer wasvisualized by VMir Viewer program which shows completeoutput in graphical manner with sequence length and scoreFigure 2 shows the graphical representation of MERS-CoVprecursor miRNAs hairpin As default setting 665 candidatehairpins (Figure 2(a)) have been identified To avoid bona fidepre-miRNAs hairpin we filtered VMir output using customsetting that is for cut-off value 60 nt minimum hairpin size220 nt maximum hairpin size and 115 minimum hairpinscore Finally 74 pre-miRNA hairpins (Figure 2(b)) wereselected as potential hairpins for further analysis

32 Prediction of Human miRNAs from Precursor miRNAsHairpin Each of the sequences of the candidate miRNA pre-cursors was searched for nucleotide similarity with all humanmicroRNAs by using humanmiRNA filter of SEARCHmenuof the miRBase database (httpwwwmirbaseorgsearchshtml) [17 27] As shown in Table 1 11 sequences wereidentified as candidatemiRNAprecursor based on significantsequence similarity with human miRNAs Human miRNAswhich show minimum 19 bp sequence similarity with can-didate miRNA precursor were selected as primary targetmiRNAs [32] For potential miRNA targets near or near toperfect alignment of those miRNAs seed region (2ndash7) werechosen that located at the 31015840 untranslated region (31015840UTR)

Hei

ght

MFEpfCentroid

25

20

15

10

5

0

(a)

Entro

py

Position

15

10

05

00

0 10 20 30 40 50 60 70

(b)

Figure 3 Mounting plot of predicted secondary structure ofprecursor miRNA hairpin Here hairpin MR268 was shown as anexample Red line green line and blue line were used to show theminimum free energy (MFE) the thermodynamic ensemble of RNA(pf) and the centroid structures respectively

of the candidate miRNA precursor Perfect complementaritybetween 31015840 untranslated region (UTR) of the mRNA and theseed region of miRNA (2ndash7 bp) is important during genesilencing Precursor miRNA hairpins were classified by MD(forward direction) andMR (reverse direction) Viral precur-sor miRNA hairpins MD5 MD17 MD110 MD157 MD186MD244 MD366 MR175 MR201 MR268 and MR282 haveshown significant identity with 13 human miRNAs Hair-pin MD110 exhibited significant sequence similarity withboth hsa-miR-3934-5p and hsa-miR-4474-5p where MD186was aligned with both hsa-miR-7974 and hsa-miR-6865-5p Other hairpins MD5 MD17 MD157 MD244 MD366MR175 MR201 MR268 and MR282 were aligned withhsa-miR-628-5p hsa-miR-6804-3p hsa-miR-4289 hsa-miR-208a-3p hsa-miR-510-3p hsa-miR-18a-3p hsa-miR-329-3phsa-miR-548ax and hsa-miR-342-3p respectively

33 Hybridization between Viral Precursor miRNAs andHuman miRNAs Effective hybridization between targethuman miRNA and precursor miRNA of MERS-CoVwas determined by the RNAhybrid tool (httpbibiserv2cebitecuni-bielefelddernahybrid) [30] RNAhybrid is atool for finding the minimum free energy hybridization of along and a short RNA and widely used for microRNA targetprediction Pairing energy or minimum free energy (MFE)indicates the stability of the hybridization For the selectionof potential miRNA the pairing energy at minus10 kcalmol wasutilized as cut-off score Effective hybridizations are shownin Box 1


Table 1 Alignments of precursor miRNAs hairpin sequences with human miRNAs

S number Hairpin Score Alignments between human microRNA and MERS CoV

1 MD5 1516 UserSeq 37 cucuagugcaaauggcagcuu

cucuaguaaauaugucagcau

17hsa-miR-628-5p 1 21

2 MD17 140 UserSeq 61 cuuuggaugugaggaagguac

cugugggugagaggcaggugc

41hsa-miR-6804-3p 2 22

3 MD110 1811

UserSeq 44 cagguguggagucugaug

cagguguggaaacugagg

61hsa-miR-3934-5p 2 19

UserSeq 71 gagucugauguugagaccaa

gugucugaucaugagacuaa

52hsa-miR-4474-5p 1 20

4 MD157 1516 UserSeq 38 gcauugugcagugguguaa

gcauugugcagggcuauca

56hsa-miR-4289 1 19

5 MD186 1511

UserSeq 52 gcuuaggcaagcagcacugcc

gcucaggagagcaucacagcc

32hsa-miR-7974 2 22

UserSeq 59 agcagcacugccccaaucu

agcaccacugcuccucucu

41hsa-miR-6856-5p 2 20

6 MD244 1273 UserSeq 1 auaagacgagugaugagcuu

auaagacgagcaaaaagcuu

20hsa-miR-208a-3p 1 20

7 MD366 1564 UserSeq 60 uccacucguagaggacucua

uccacucuuagagguuucaa

41hsa-miR-510-3p 2 21

8 MR175 1409 UserSeq 5 acuggccugaaagcuccuucuug

acugcccuaagugcuccuucugg

27hsa-miR-18a-3p 1 23

9 MR201 1488 UserSeq 31 aaugagguuacacagguaagu

aaagagguuaaccaggugugu

11hsa-miR-329-3p 2 22

10 MR268 137 UserSeq 41 uggcaacgccggaauuaguuc

uggcaaaaccgcaauuacuuc

21hsa-miR-548ax 2 22

11 MR282 1636 UserSeq 50 acgggugcgagugcgcugaguga

acgggugcgauuucugugugaga

28hsa-miR-342-3p 1 23

34 Prediction of Secondary Structure of miRNA PrecursorThe RNAfold web server (httprnatbiunivieacatcgi-binRNAfoldcgi) was used to predict the secondary structure ofpre-miRNA (shown in Figures 3 and 4) [31] Only defaultparameters were used The RNAfold program was usedto predict the most stable secondary structure of MERS-CoVHairpin sequencesThe sequence applied for predictionanalysis included pre-miRNA about 200 bp upstream andabout 100 bp downstream flanking sequences at each endof the precursor [27] In all cases folding structures withcentroid were depicted

4 Discussion

MicroRNAs (miRNAs) are genomically encoded a class ofsmall noncoding RNAs (sim22 nt) which normally function asnegative regulators of target mRNA expression at the stage ofposttranscriptional level [12ndash14 33]The perfect complemen-tarity between 31015840 untranslated region (UTR) of the mRNAand the seed region of miRNA (2ndash7 bp) is thought to be

sufficient for effective cleavage but imperfect complementar-ity may block translation [33] There is increasing evidencesuggesting that miRNAs play critical roles in many key bio-logical processes such as cell growth tissue differentiationcell proliferation embryonic development cell proliferationand apoptosis [34] Aberrant expression of miRNAs includ-ingmutation dysfunction and dysregulation ofmiRNAs andtheir targets may result in various diseases such as cancers[34 35] cardiovascular disease [36 37] schizophrenia [3839] psoriasis [40] and primary muscular disorders [41] Inthe meantime miRNAs have been great interest in utilizingmiRNAs as a nonpharmaceutical approach to treat numer-ous diseases including HIV-1 HSV Dengue Influenza andhepatitis C (HCV)

The use of miRNAs as anti-HCV treatment demonstratedpromising efficacy and safety results in an early stage trialJanssen et al [26] reported that locking the liver-expressedmicroRNA-122 (miR-122) led to dose-dependent and persis-tent decline in HCV By utilizing a series of bioinformaticstools we predict 13 potential cellular miRNAs targeting


(A) MD5 amp hsa-miR-628-5p (MFE minus235 kcalmol) (B) MD17 amp hsa-miR-6804-3p (MFE minus233 kcalmol)

MD5 5 1015840 G CA GCAGCUUG C 3 1015840 target 5 1015840 A U A U A A 3 1015840

CUCUAGUG AAUG GUUGGCA CU UGG UG GAGG AGGU

GAGAUCAU UUAU CAGUCGU GA ACC AC CUCC UCCA

miRNA 3 1015840 G A A 5 1015840 miRNA 3 1015840 C C U G CGC 5 1015840

(C) MD110 amp hsa-miR-3934-5p (MFE minus171 kcalmol) (D) MD110 amp hsa-miR-4474-5p (MFE minus267 kcalmol)

target 5 1015840 A UC UG C 3 1015840 target 5 1015840 A GU C 3 1015840

CUUA UCUAC CU GUCUGAU UGAGAC

GAGU AGGUG GA CAGACUA ACUCUG

miRNA 3 1015840 GACG CAA UG CU 5 1015840 miRNA 3 1015840 ACA GU AUU 5 1015840

(E) MD157amp hsa-miR-4289 (MFE minus217 kcalmol) (F) MD244 amp hsa-miR-208a-3p (MFE minus194 kcalmol)

target 5 1015840 C U A G 3 1015840 target 5 1015840 G GC GAA A 3 1015840

G UAGC UUGUGCAGUG AUGAGCUUU GU UCUUA

C AUCG GACGUGUUAC UGUUCGAAA CG AGAAU

miRNA 3 1015840 A U G G 5 1015840 miRNA 3 1015840 AA AGC A 5 1015840

(G) MD186 amp hsa-miR-7974 (MFE minus311 kcalmol) (H) MD186 amp hsa-miR-6856-5p (MFE minus298 kcalmol)

target 5 1015840 C CA G U C 3 1015840 target 5 1015840 G A G C AA A 3 1015840

GCUUAGG AGCA CAC GCC GC AGCA CACUGC CC UCU

CGAGUCC UCGU GUG CGG UG UCGU GUGACG GG AGA

miRNA 3 1015840 CC UC A U A 5 1015840 miRNA 3 1015840 GG G A AG A 5 1015840

(I) MD366 amp hsa-miR-510-3p (MFE minus264 kcalmol) (J) MR175 amp hsa-miR-18a-3p (MFE minus209 kcalmol)

target 5 1015840 G G A 3 1015840 target 5 1015840 G CAA CA G 3 1015840

UCCACUC UAGAGG UAGA GAGCGCU CAGU

AGGUGAG AUCUCC GUCU CUCGUGA GUCA

miRNA 3 1015840 A AAAGUUA 5 1015840 miRNA 3 1015840 G UC AUCCC 5 1015840

(K) MR201 amp has-miR-329-3p (minus224 kcalmol) (L) MR268 amp hsa-miR-548ax (MFE minus285 kcalmol)

target 5 1015840 U CA AA A 3 1015840 target 5 1015840 A C G G C 3 1015840

GAGGUUA CAGGU GU UGGCAA GCCG AAUUA UUC

CUCCAAU GUCCA CA ACCGUU UGGC UUAAU AAG

miRNA 3 1015840 UUU UG CA A 5 1015840 miRNA 3 1015840 U G G A 5 1015840

(M) MR282 amp hsa-miR-342-3p (MFE minus335 kcalmol)

target 5 1015840 G GUGCG A U 3 1015840

ACGGGUGCGA CUG GUGA

UGCCCACGCU GAC CACU

miRNA 3 1015840 AAA A CU 5 1015840

Box 1 Hybridization between microRNA and viral RNA using RNA hybrid program The program finds the energetically most favorablehybridization sites of a miRNA in a large hairpin of viral RNA

the MERS-CoV virus while except 3 miRNAs all otherpredicted humanmiRNAs functions yet to be discovered Butbased on our computational investigation we hypothesizethat those miRNAs may have significant role to know hostpathogen interaction in order to develop potential therapeu-tics Thus far the 10 miRNAs that is hsa-miR-6804-3p hsa-miR-4289 hsa-miR-208a-3p hsa-miR-510-3p hsa-miR-329-3p hsa-miR-548ax hsa-miR-3934-5p hsa-miR-4474-5p hsa-miR-7974 and hsa-miR-6865-5p do not have any knownfunction in human and other animals

41 Roles of hsa-miR-628-5p hsa-miR-18a-3p and hsa-miR-332-3p in Humans Those 3 miRNAs have shown significantsequence identity with MERS-CoV genome where seed

region of hsa-miR-628-5p and hsa-miR-332-3p showed per-fect identity with 31015840 untranslated region of viral mRNA Ithas been reported that hsa-miR-628-5p associated with mostcommon brain cancer glioma and acted as protective factorswhere their expression decreased gradually during gliomaprogression [42] Functional analysis of thismiRNA indicatesthat they have critical roles in cell cycle and cell proliferationin glioblastoma malignant progression where has-miR-628-5p exhibited dominant regulatory activities [42]

Also hsa-miR-18a is unregulated in basal cell carcinoma(BCC) of the skin compared with non-lesional skin [43] Fur-ther study indicates that along with other miRNAs hsa-miR-18a target genes were predominantly involved in the regula-tion of cell proliferation differentiation and adhesion during


MD5 (minus2400Kcalmol) MD17 (minus2300Kcalmol) MD110 (minus3320Kcalmol)

MD157 (minus2420Kcalmol) MD186 (minus2470Kcalmol) MD244 (minus1670Kcalmol) MD366 (minus3170Kcalmol)

MR175 (minus1910Kcalmol) MR201 (minus1640Kcalmol) MR268 (minus2090Kcalmol) MR282 (minus2620Kcalmol)

Figure 4 Predicated secondary structure of potential hairpins candidate of MERS-CoV Only centroid structures were depicted

the process of malignant transformation [44] In additionhsa-miR-332-3p is thought to associate with idiopathic priondisease and it has been proposed that the upregulation ofhsa-miR-342-3p may be a general phenomenon in late stageprion disease andmight be used as a novel marker for animaland Human Transmissible Spongiform Encephalopathies(human TSEs) [45]

5 Conclusion

By utilizing a series of bioinformatics tools we predict thecandidate potential miRNA targeting MERS-CoV The resultsuggested the miRNAs from MD5 MD17 MD110 MD157MD186 MD244 MD366 MR175 MR201 MR268 andMR282 hairpins would be best candidate for targeting human


cellular miRNAs The utility of those 13 miRNAs that ishsa-miR-628-5p hsa-miR-6804-3p hsa-miR-4289 hsa-miR-208a-3p hsa-miR-510-3p hsa-miR-18a-3p hsa-miR-329-3phsa-miR-548ax hsa-miR-3934-5p hsa-miR-4474-5p hsa-miR-7974 hsa-miR-6865-5p and hsa-miR-342-3p can beutilized as antiviral therapeutics against MERS-CoV infec-tion However further in vitro study should be performed inorder to assess the inhibition influence on viral replication bythe effect of selected human miRNAs

Abbreviations

MERS-CoV Middle East Respiratory SyndromeCoronavirus

miRNA MicroRNAhsa-miR Human microRNA

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

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[2] N Zhang S Jiang and L Du ldquoCurrent advancements andpotential strategies in the development of MERS-CoV vac-cinesrdquoExpert Review of Vaccines vol 13 no 6 pp 761ndash774 2014

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[4] L M Gardner and C R MacIntyre ldquoUnanswered ques-tions about the Middle East respiratory syndrome coronavirus(MERS-CoV)rdquo BMC Research Notes vol 7 no 1 p 358 2014

[5] E I Azhar S A El-Kafrawy S A Farraj et al ldquoEvidence forcamel-to-human transmission of MERS-coronavirusrdquoTheNewEngland Journal of Medicine vol 370 pp 2499ndash2505 2014

[6] N vanDoremalen K LMiazgowicz SMilne-Price et al ldquoHostspecies restriction of middle east respiratory syndrome coron-avirus through its receptor dipeptidyl peptidase 4rdquo Journal ofVirology 2014

[7] A Lundin R Dijkman T Bergstrom et al ldquoTargeting mem-brane-bound viral RNA synthesis reveals potent inhibition ofdiverse coronaviruses including the middle East respiratorysyndrome virusrdquo PLoS Pathogens vol 10 no 5 Article IDe1004166 2014

[8] A H de Wilde V S Raj D Oudshoorn et al ldquoMERS-coro-navirus replication induces severe in vitro cytopathology and isstrongly inhibited by cyclosporin A or interferon-120572 treatmentrdquoJournal of General Virology vol 94 part 8 pp 1749ndash1760 2013

[9] A Eulalio E Huntzinger and E Izaurralde ldquoGetting to theRoot of miRNA-Mediated Gene Silencingrdquo Cell vol 132 no 1pp 9ndash14 2008

[10] M T McManus and P A Sharp ldquoGene silencing in mammalsby small interfering RNAsrdquo Nature Reviews Genetics vol 3 no10 pp 737ndash747 2002

[11] I Ryu J H Park S An O S Kwon and S K Jang ldquoeIF4GIfacilitates the MicroRNA-mediated gene silencingrdquo PLoS ONEvol 8 no 2 Article ID e55725 2013

[12] V Ambros ldquoThe functions of animal microRNAsrdquo Nature vol431 no 7006 pp 350ndash355 2004

[13] D P Bartel ldquoMicroRNAs genomics biogenesis mechanismand functionrdquo Cell vol 116 no 2 pp 281ndash297 2004

[14] C Kwon Z Han E N Olson and D Srivastava ldquoMicroRNA1influences cardiac differentiation in Drosophila and regulatesNotch signalingrdquo Proceedings of the National Academy of Sci-ences of the United States of America vol 102 no 52 pp 18986ndash18991 2005

[15] V Ambros ldquomicroRNAs tiny regulators with great potentialrdquoCell vol 107 no 7 pp 823ndash826 2001

[16] J C Carrington and V Ambros ldquoRole of microRNAs in plantand animal developmentrdquo Science vol 301 no 5631 pp 336ndash338 2003

[17] A Kozomara and S Griffiths-Jones ldquoMiRBase annotating highconfidence microRNAs using deep sequencing datardquo NucleicAcids Research vol 42 no 1 pp D68ndashD73 2014

[18] C S Sullivan and D Ganem ldquoMicroRNAs and viral infectionrdquoMolecular Cell vol 20 no 1 pp 3ndash7 2005

[19] T Du and P D Zamore ldquomicroPrimer the biogenesis andfunction ofmicroRNArdquoDevelopment vol 132 no 21 pp 4645ndash4652 2005

[20] M L Yeung Y Bennasser S Y Le and K T Jeang ldquosiRNAmiRNA and HIV promises and challengesrdquo Cell Research vol15 no 11-12 pp 935ndash946 2005

[21] H Zhang Z Li Y Li et al ldquoA computational method forpredicting regulation of human microRNAs on the influenzavirus genomerdquo BMC Systems Biology vol 7 supplement 2 pS3 2013

[22] V R Sanghvi and L F Steel ldquoRNA silencing as a cellulardefense against HIV-1 infection progress and issuesrdquo TheFASEB Journal vol 26 no 10 pp 3937ndash3945 2012

[23] Z Wu Y Zhu D M Bisaro and D S Parris ldquoHerpes simplexvirus type 1 suppresses RNA-induced gene silencing in mam-malian cellsrdquo Journal of Virology vol 83 no 13 pp 6652ndash66632009

[24] M Hussain and S Asgari ldquoMicroRNA-like viral small RNAfrom Dengue virus 2 autoregulates its replication in mosquitocellsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 111 no 7 pp 2746ndash2751 2014

[25] IM Pedersen G Cheng SWieland et al ldquoInterferonmodula-tion of cellular microRNAs as an antiviral mechanismrdquo Naturevol 449 no 7164 pp 919ndash922 2007

[26] H L Janssen H W Reesink E J Lawitz et al ldquoTreatment ofHCV infection by targeting microRNArdquo The New EnglandJournal of Medicine vol 368 no 18 pp 1685ndash1694 2013

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[28] A Grundhoff C S Sullivan and D Ganem ldquoA combinedcomputational and microarray-based approach identifies novelmicroRNAs encoded by human gamma-herpesvirusesrdquo RNAvol 12 no 5 pp 733ndash750 2006

[29] C S Sullivan andA Grundhoff ldquoIdentification of viral microR-NAsrdquoMethods in Enzymology vol 427 pp 3ndash23 2007

[30] J Kruger and M Rehmsmeier ldquoRNAhybrid microRNA targetprediction easy fast and flexiblerdquoNucleic Acids Research vol 34pp W451ndashW454 2006

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[32] A Khokhar S Noorali M Sheraz et al ldquoComputational analy-sis to predict functional role of hsa-miR-3065-3p as an antiviraltherapeutic agent for treatment of triple infectionsHCVHIV-1and HBVrdquo The Libyan Journal of Medicine vol 7 Article ID19774 2012

[33] M L Casal D M Dambach T Meister P F Jezyk D F Pat-terson and P S Henthorn ldquoFamilial glomerulonephropathy inthe BullmastiffrdquoVeterinary Pathology vol 41 no 4 pp 319ndash3252004

[34] A Esquela-Kerscher and F J Slack ldquoOncomirsmdashmicroRNAswith a role in cancerrdquo Nature Reviews Cancer vol 6 no 4 pp259ndash269 2006

[35] M Serban I Ghiorghiu I Craciunescu et al ldquoSpontaneousecho contrast of unexpected etiologyrdquo European Journal ofEchocardiography vol 7 no 3 pp 257ndash259 2006

[36] M V Latronico D Catalucci and G Condorelli ldquoEmergingrole of microRNAs in cardiovascular biologyrdquo CirculationResearch vol 101 no 12 pp 1225ndash1236 2007

[37] E vanRooij L B Sutherland XQi J A Richardson J Hill andE N Olson ldquoControl of stress-dependent cardiac growth andgene expression by a microRNArdquo Science vol 316 no 5824 pp575ndash579 2007

[38] T Hansen L Olsen M Lindow et al ldquoBrain expressed mic-roRNAs implicated in schizophrenia etiologyrdquo PLoS ONE vol2 no 9 article e873 2007

[39] D O Perkins C D Jeffries L F Jarskog et al ldquomicroRNAexpression in the prefrontal cortex of individuals with schiz-ophrenia and schizoaffective disorderrdquo Genome Biology vol 8no 2 article R27 2007

[40] E Sonkoly T Wei P C J Janson et al ldquoMicroRNAs novel reg-ulators involved in the pathogenesis of psoriasisrdquo PLoS ONEvol 2 no 7 Article ID e0000610 2007

[41] Y S Al-FaiyzMM El-Garawany F N Assubaie andM A Al-Eed ldquoImpact of phosphate fertilizer on cadmium accumulationin soil and vegetable cropsrdquo Bulletin of Environmental Contam-ination and Toxicology vol 78 no 5 pp 358ndash362 2007

[42] Y Li J Xu H Chen et al ldquoComprehensive analysis of the func-tional microRNA-mRNA regulatory network identifies miRNAsignatures associated with glioma malignant progressionrdquoNucleic Acids Research vol 41 no 22 p e203 2013

[43] M Sand M Skrygan D Sand et al ldquoExpression of microRNAsin basal cell carcinomardquo British Journal of Dermatology vol 167no 4 pp 847ndash855 2012

[44] F-M Cui J-X Li Q Chen et al ldquoRadon-induced alterations inmicro-RNA expression profiles in transformed BEAS2B cellsrdquoJournal of Toxicology and Environmental Health A vol 76 no2 pp 107ndash119 2013

[45] J Montag R Hitt L Opitz W J Schulz-Schaeffer G Hun-smann and D Motzkus ldquoUpregulation of miRNA hsa-miR-342-3p in experimental and idiopathic prion diseaserdquoMolecularNeurodegeneration vol 4 no 1 article 36 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Virolog y

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


MERS CoV (Strain Riad) complete genome

Hairpin like structure screen by VMir

74 miRNAs precursor candidate

Screen with all human miRNA by miRbase search

11 pre-miRNAs aligned with 13 human miRNAs

RNA hybrid web server to effective hybridization

RNAfold to predict secondary structure

Figure 1 Schematic representation of human miRNA prediction on MERS-CoV viral genome [27]

miRNAs are genomically encoded small noncoding RNAmolecule generally 19ndash26 base pairs in length which regulateposttranscriptional level genes expression [12ndash14] It is welldocumented that some plants animals and viruses encodethe miRNAs to regulate their diverse biological or physio-logical processes including development apoptosis tumor-ogenesis proliferation stress response and fat metabolism[15 16] Thus 30 424 mature microRNAs have been identifiedfrom 206 species where 2578miRNAs are encoded by humangenome [17] Virus encodedmiRNAs are unique because theyregulate not only their own gene expression but also their hostgene expression [18]

miRNA genes are transcribed by RNA polymerase IIand formed primary miRNA in nucleus Then primary miR-NAs cleaved into 60ndash90 base-pair-long hairpin intermediateknown as pre-miRNA by enzymatic activity of the RNaseIII ribonuclease Dicer [18ndash20] Pre-miRNAs are bound andexported from nucleus to cytoplasm by the action of enzymeexportin-5 and Ran (RAs-related Nuclear protein) [19] Inthe cytoplasm the pre-miRNAs are further cleaved by RNaseIII ribonuclease Dicer into a double stranded RNA knownas duplex mature RNA [19] Guided stand (active stand) ofduplex RNA is loaded to RNA-induced silencing complex(RISC) which targets messenger RNA to degrade or represstranslational activity [18] Perfect complementarity between31015840 untranslated region (UTR) of the mRNA and the seedregion of miRNA (2ndash7 bp) is sufficient result in cleavage butimperfect complementarity may block translation [18 21]Some recent study suggests that miRNA is being exploredas antiviral defense against several diseases including HIV-1 [22] HSV [23] Dengue [24] Influenza [21] and hepatitisC (HCV) [25] It has been reported that the use of miRNAsas an anti-HCV treatment demonstrated promising efficacy

and safety results in an early stage trial [26] In this study wecomputationally identified some potential targets of humanmicroRNA on Middle East Respiratory Syndrome Coron-avirus (MERS-CoV) genome Our study may help to betterunderstand host pathogen interaction as well as to developnew antiviral therapy against MERS-CoV

2 Materials and Methods

TheMERS-CoVmiRNA prediction was carried out using thecomplete genome sequence of MERS-CoV (GB KJ1569521)obtained from the National Center for Biotechnology Infor-mation (NCBI) Figure 1 shows a flowchart of the compu-tational prediction process [27] Briefly the viral genomewas scanned for hairpin-structured miRNA precursors usinga VMir Analyzer program [28 29] VMir is an ab initioprediction program which was designed specifically to iden-tify pre-miRNA in viral genome The scanned hairpins werevisualized in VMir viewer where 74 sequences with potentialhairpin like structures were extracted as candidate miRNAprecursor Each of the sequences of the candidate miRNAprecursors was searched for nucleotide similarity with allhuman microRNAs by using SEARCHmenu of the miRBasedatabase (httpwwwmirbaseorgsearchshtml) [17]

Then 11 sequences were identified as candidate miRNAprecursor based on significant sequence similarity withhuman miRNAs The hybridization between the viral pre-cursor miRNAs and complementary template of the poten-tial human miRNAs were analyzed by RNA hybrid webserver (httpbibiserv2cebitecuni-bielefelddernahybrid)[30] Finally the RNAfold web server (httprnatbiunivieacatcgi-binRNAfoldcgi) was used to predict the secondarystructure of pre-miRNA [31]


225

200

150

100

50

0

1 5000 10000 15000 20000 25000 30000

(a)

200

150

100

50

0

1 5000 10000 15000 20000 25000 30000

(b)


3 Result



Hei

ght

MFEpfCentroid

25

20

15

10

5

0

(a)

Entro

py

Position

15

10

05

00

0 10 20 30 40 50 60 70

(b)









17hsa-miR-628-5p 1 21



41hsa-miR-6804-3p 2 22

3 MD110 1811


cagguguggaaacugagg

61hsa-miR-3934-5p 2 19



52hsa-miR-4474-5p 1 20


gcauugugcagggcuauca

56hsa-miR-4289 1 19

5 MD186 1511



32hsa-miR-7974 2 22


agcaccacugcuccucucu

41hsa-miR-6856-5p 2 20



20hsa-miR-208a-3p 1 20



41hsa-miR-510-3p 2 21



27hsa-miR-18a-3p 1 23



11hsa-miR-329-3p 2 22






28hsa-miR-342-3p 1 23


4 Discussion




































target 5 1015840 G GUGCG A U 3 1015840

ACGGGUGCGA CUG GUGA

UGCCCACGCU GAC CACU

miRNA 3 1015840 AAA A CU 5 1015840












5 Conclusion




Abbreviations





References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


225

200

150

100

50

0

1 5000 10000 15000 20000 25000 30000

(a)

200

150

100

50

0

1 5000 10000 15000 20000 25000 30000

(b)


3 Result



Hei

ght

MFEpfCentroid

25

20

15

10

5

0

(a)

Entro

py

Position

15

10

05

00

0 10 20 30 40 50 60 70

(b)









17hsa-miR-628-5p 1 21



41hsa-miR-6804-3p 2 22

3 MD110 1811


cagguguggaaacugagg

61hsa-miR-3934-5p 2 19



52hsa-miR-4474-5p 1 20


gcauugugcagggcuauca

56hsa-miR-4289 1 19

5 MD186 1511



32hsa-miR-7974 2 22


agcaccacugcuccucucu

41hsa-miR-6856-5p 2 20



20hsa-miR-208a-3p 1 20



41hsa-miR-510-3p 2 21



27hsa-miR-18a-3p 1 23



11hsa-miR-329-3p 2 22






28hsa-miR-342-3p 1 23


4 Discussion




































target 5 1015840 G GUGCG A U 3 1015840

ACGGGUGCGA CUG GUGA

UGCCCACGCU GAC CACU

miRNA 3 1015840 AAA A CU 5 1015840












5 Conclusion




Abbreviations





References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






17hsa-miR-628-5p 1 21



41hsa-miR-6804-3p 2 22

3 MD110 1811


cagguguggaaacugagg

61hsa-miR-3934-5p 2 19



52hsa-miR-4474-5p 1 20


gcauugugcagggcuauca

56hsa-miR-4289 1 19

5 MD186 1511



32hsa-miR-7974 2 22


agcaccacugcuccucucu

41hsa-miR-6856-5p 2 20



20hsa-miR-208a-3p 1 20



41hsa-miR-510-3p 2 21



27hsa-miR-18a-3p 1 23



11hsa-miR-329-3p 2 22






28hsa-miR-342-3p 1 23


4 Discussion




































target 5 1015840 G GUGCG A U 3 1015840

ACGGGUGCGA CUG GUGA

UGCCCACGCU GAC CACU

miRNA 3 1015840 AAA A CU 5 1015840












5 Conclusion




Abbreviations





References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































target 5 1015840 G GUGCG A U 3 1015840

ACGGGUGCGA CUG GUGA

UGCCCACGCU GAC CACU

miRNA 3 1015840 AAA A CU 5 1015840












5 Conclusion




Abbreviations





References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







5 Conclusion




Abbreviations





References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Abbreviations





References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article a computational approach for predicting...

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